Atherosclerosis is a condition characterized by irregularly distributed lipid deposits in the intima of large and medium-sized arteries. The deposits provoke fibrosis and calcification. Disorders involving atherosclerosis, such as coronary artery, cerebrovascular and peripheral vascular disease are the most common cause of death in the Western hemisphere. The World Health Organization (WHO) suggests that atherosclerosis-related diseases will be the leading cause of mortality in the world by the year 2020.
Various procedures are routinely used to treat atherosclerosis-related disorders such as bypass surgery and angioplasty. Although these are generally effective, they are highly invasive and complex to perform. In addition, they often do not succeed when the diseased region includes ischemic tissue, such as in the case of the blockage of an arterial tree or of a bypass graft. In such cases, an alternative, often concurrent, treatment is to stimulate the generation of new blood vessels to replace those damaged by the disease. Two types of new blood vessel formation occur naturally in adult human beings, recapitulated arteriogenesis and angiogenesis.
Recapitulated arteriogenesis involves the transformation of pre-existing arterioles into small muscular arteries. Angiogenesis is the sprouting of new blood vessels from existing ones. Angiogenesis occurs both in healthy individuals and those suffering from pathological conditions. An example of the former is the female reproductive cycle where angiogenesis occurs during the rebuilding of the lining of the uterus. An example of the latter is cancer where new blood vessels are formed in and around a growing tumor.
The angiogenic process is regulated by biomechanical and biochemical stimuli and occurs in three major stages. In the first stage, termed initiation, the connection between endothelial cells (EC) and the surrounding tissue is severed. In the second stage EC proliferate and invade the ischemic tissue, which results in formation of EC sprouts. In the third stage, the newly formed EC sprouts mature into functional blood vessels. Maturation of the blood vessels involves recruitment of cells that surround the endothelial cells such as pericytes in the capillaries, smooth muscle cells in larger vessels and cardiac myocytes in the heart. These cells provide structural support to the forming vessels and modulate their function.
The establishment and remodeling of blood vessels is controlled by paracrine signals, many of which are mediated by protein ligands that modulate the activity of transmembrane tyrosine kinase receptors. Among these ligands and receptors are vascular endothelial growth factor (VEGF) and its receptor families (VEGFR1 and VEGFR2), Angiopoietin 1 and 2 (Ang-1 and Ang-2) and their receptor (Tie 2), acidic and basic fibroblast growth factor (aFGF, bFGF), platelet derived growth factor (PDGF), transforming growth factors α and β (TGF-α, TGF-β) and tumor necrosis factor α (TNF-α).
The role of VEGF and its receptors in preliminary stages of angiogenesis has been clearly demonstrated using VEGF receptor null heterozygous animals (Hanahan, D., Science, 1997, 277:48-50; Ferrara, N., Carver-Moore, K., Chen, H., et al., Nature, 1996, 380:439-42; Shalaby, F., Rossant, J., Yamaguchi, T. P., et al., Nature, 1995, 376:62-66). These animals, which do not survive the early stages of embryogenesis, either do not produce EC when heterozygous for the VEGFR1 receptor, or fail to form vessels when heterozygous for the VEGFR2 receptor. Studies in which the Tie2 receptor or its ligands Ang-1 and Ang-2 were disrupted demonstrated that although EC formed a tube, periendothelial cells were not recruited (Fong, G. H., Rossant, J., Gertenstein, M., et al., Nature, 376:66-70; Dumont, D. J., Gradwhol, G., Fong, G-H., et al., Genes Dev., 1994, 8:1897-1903; Sato, T. N., Tzoawa, Y., Deutsch, U., et al., Nature, 1995, 376:70-74; Suri, C., Jones, P. F., Patan, S., et al., Cell, 1996, 87:1171-80; Maisonpierre, P. C., Suri, C., Jones, P. F., et al., Science, 1997, 277:55-60). A similar phenotype was observed in animals lacking PDGF-B, TGF-β and tissue factor (Leveen, P., Pekny, M., Gebre-Medhin, S., et al., Genes Dev., 1994, 8:1875-87; Carmeliet, P., Mackman, N., Moons, L., et al., Nature, 1996, 383:73-75; Lindhal, P., Johansson, B. R., Leveen, P., Hbetsholtz, C., Science, 1997, 277:242-245) suggesting that the binding of angiopoietin to its receptor may lead to the secretion of these factors from the endothelium. Other studies have suggested that VEGF is responsible for the early stage of angiogenesis, which is characterized by disintegration of EC and leakage of plasma components (Nat. Med., 2000, 6:131-2, 6:460-3). There are studies that suggest that Ang-1 regulates the maturation of newly formed blood vessels, while other studies suggest that the binding of Ang-2 to Tie2 plays a role in the regression of existing vessels (Suri, C., Jones, P. F., Patan, S, et al., Cell, 1996,87:1171-80).
Whether or not angiogenesis occurs in a particular situation is determined by changes in the local equilibrium among angiogenic modulators, i.e., stimulatory and inhibitory factors. In this regard, gene therapy, the insertion into cells of genes that express the modulators has received a great deal of attention. For example, gene therapy has been examined both in vitro and in vivo as a means for inhibiting smooth muscle cell proliferation following angioplasty or bypass surgery and for inducing angiogenesis by enhancement of EC cell proliferation. Naked-DNA and recombinant adenoviral vectors encoding VEGF165 and VEGF121 have been used to transfer genes in vivo to human patients suffering from ischemic and peripheral vascular disease to genetically modify endogenous vascular cells to express angiogenic factors.
Co-administration of VEGF and Ang-1 encoding vectors in an animal model has been shown to enhance the development of collateral vessels. However, as with the above methods, this approach would be expected to be of limited therapeutic utility in patient's with ischemic tissues and damaged organs because of a shortage of healthy cells to infect.
What is needed is a safe, effective method for inducing angiogenesis in tissue, in particular ischemic tissue. The present invention provides such a method using vascular cells genetically altered to over-express angiogenic and vascular maturation factors.